CRAF (IC50 = 0.072 nM); Braf (IC50 = 0.21 nM); ARAF (IC50 = 6.4 nM); p38α (IC50 = 2.1 μM); Abl1 (IC50 = 4.9 μM)

LXH254 (Compound A) is an adenosine triphosphate (ATP)-competitive inhibitor of BRAF (also referred to herein as b-RAF or b-Raf) and CRAF (also referred to herein as c-RAF or c- Raf) protein kinases. LXH254 is also referred to as a C-RAF/c-Raf kinase inhibitor and as a c-RAF (or CRAF) inhibitor throughout the present disclosure. In cell-based assays, LXH254 has proven to have anti-proliferative effects in cell lines with a variety of mutations that stimulate MAPK signaling. Additionally, LXH254 is a Type 2 ATP-competitive inhibitor of both B-Raf and C-Raf that maintains the kinase pocket in an inactive conformation, reducing the paradoxical activation seen with many B-Raf inhibitors and inhibiting mutant RAS-driven signaling and cell proliferation[1].
LXH254 (0–10 µM, 1 h) inhibits monomeric and dimeric RAF and encourages the formation of RAF dimers[2].
The ability of LXH254 to inhibit MAPK signaling driven by ARAF is decreased, and it has also been shown that when CRAF expression is absent, ARAF's contribution to MAPK signaling increases[2].
LXH254 shows more sensitivity when cells lack ARAF[2].

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Researchers describe an unexpected paralog selectivity of Naporafenib (LXH-254), which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib.

In a number of KRAS-mutant models, including the NSCLC-derived Calu-6 (KRAS Q61K) and NCI-H358 (KRAS G12C), treatment with LXH254 (Compound A) results in tumor regression. Numerous MAPK-driven human cancer cell lines and xenograft tumors that represent model tumors with human lesions in the KRAS, NRAS, and BRAF oncogenes show efficacy for LXH254[1].
In models with BRAF mutations, whether they are present alone or in combination with activated NRAS or KRAS, LXH254 exhibits significant antitumor activity, and RAS mutants deficient in ARAF are more responsive to LXH254[2].

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RAS mutants lacking ARAF are more sensitive to Naporafenib (LXH-254) in vivo [2]
We next determined the effect of ARAF ablation on sensitivity to MAPK inhibitors in vivo. Tumors formed from parental and ARAF-deleted variants of the HCT 116, MIA PaCa-2, and MEL-JUSO models were treated with vehicle, 0.3 mg/kg every day trametinib, or 50 mg/kg twice a day Naporafenib (LXH-254). Doses of trametinib and LXH254 were selected that matched AUC values for the approved dose of trametinib (2 mg, twice a day) or the recommended phase II dose of LXH254 (400 mg, twice a day). ARAF-deleted xenografts either grew with similar kinetics (HCT 116 cells) or slightly slower (MIA PaCa-2 and MEL-JUSO) than their parental counterparts, indicating that ARAF ablation has at most modest effects on the fitness of these models. In all parental models, LXH254 and trametinib exerted similar effects on tumor growth, resulting in a slow growth phenotype, which was most pronounced in MEL-JUSO cells (Fig. 7A–C). Treatment with LXH254 in the ARAF knockouts led to complete regression of HCT 116 and MEL-JUSO and near-complete regression of MIA PaCa-2 xenografts. Several tumors from each KRAS-mutant models regrew after prolonged treatment, including three of six from the HCT 116 and five of six of the MIA PaCa-2 tumors (Supplementary Fig. S4A–S4C). Initial analysis of these variants by Western blot analysis suggested that at least two tumors (MIA PaCa-2, M2, and M5) became resistant due to RAF pathway reactivation, with reactivation in one case potentially attributable to restoration of ARAF expression (M5). Lack of clear pathway reactivation in the other six tumors suggested resistance occurred via a MAPK bypass mechanism (Supplementary Fig. S4D). Strikingly, two of three of the regressed HCT 116 tumors failed to regrow after cessation of drug treatment consistent with complete eradication of the tumor (Supplementary Fig. S4A). In contrast, variants lacking ARAF expression had similar sensitivities to trametinib as parental MIA PaCa-2 cells.
These data indicate that RAS-driven activation of BRAF and CRAF is inhibited sufficiently by Naporafenib (LXH-254) to eradicate tumors, and conversely that poor ARAF inhibition is a critical contributor to the relative insensitivity of RAS-mutant models to LXH254. Accordingly, we tested whether cotreatment of tumors with LXH254 and low doses of trametinib, aimed at inhibiting residual ARAF-driven signaling, improved antitumor effects in MIA PaC-2 cells. Combining LXH254 with either 50% (0.3 mg/kg, every 2 days) or 10% (0.03 mg/kg once a day) doses of trametinib improved efficacy relative to both LXH254 and a full dose of trametinib (0.3 mg/kg, twice a day), although efficacy was inferior to that seen for single-agent LXH254 in cells lacking ARAF (Fig. 7D), and tumors regrew over time (data not shown). Thus, combining LXH254 with even small amounts of an MEK inhibitor can result in significant antitumor effects.

RAF in vitro enzyme assays [2]
Biochemical inhibition of ARAF, BRAF, and CRAF, shown in Fig. 1A, was performed as described for RAF709 in Shao and colleagues. In brief, the catalytically inactive MEK1K97R variant was used as a substrate for either full-length BRAF, full-length activated CRAF (Y340E/Y341E variant), or a full-length N-terminal GST-ARAF fusion. In all cases, Naporafenib (LXH-254) was preincubated with CRAF/BRAF/ARAF for 30 minutes prior to substrate addition/reaction initiation. Biochemical activity of Naporafenib (LXH-254) in the extended panel of kinases, shown in Supplementary Table S1, was determined as described in Wylie and colleagues.

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Structural modeling [2]
The in-house X-ray structure (PDB entry: 6N0P) of Naporafenib (LXH-254) bound to BRAF was used in conjunction with an X-ray structure of CRAF (PDB entry: 3OMV) to build a homology model of ARAF with Naporafenib (LXH-254) bound in the molecular operating environment. This model was extensively refined with explicated solvent molecular dynamics simulations at 300 K, 1 atm, AM1BCC/ELF charges, TIP3P water, and ff14SB force field with the PARM@FROSST extension within the AMBER suite of simulation tools. Ten-nanosecond simulations for each system were run and time average structures were extracted.

Cell-based kinase assays [2]
In-cell kinase selectivity profiling was performed by KiNativ. Briefly, HCT 116 cells were treated with Naporafenib (LXH-254) for 2 hours at 10 μmol/L, and then lysates were processed, probe labeled, and analyzed by LC/MS-MS, as described previously. For immunoprecipitate (IP)-kinase assays, cells were cultured in compound for 4–24 hours prior to protein immunoprecipitation, as described above. IPs were washed once with IP lysis buffer and twice with 1 × Kinase Buffer, and protein-bound beads were incubated in 2 × kinase buffer with 250 μmol/L ATP and 0.5 μg MEK1 or MEK1-K97M for 30 minutes at 30°C. Beads were then washed three times with IP lysis buffer and prepared for immunoblotting as described above.

Outbred athymic (nu/nu) female mice and SCID Beige mice; BRAF-, NRAS-, and KRAS-mutant xenograft models, as well as a RAS/RAF wild-type model[2]
100 mg/kg
Orally, daily

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Cell culture and in vivo efficacy [2]
All cell lines were shown to be free of Mycoplasma species and murine viruses in the IMPACT VIII PCR Assay Panel (IDEXX RADIL, IDEXX Laboratories Inc). In all cases, cells were harvested at 80%–95% confluence with 0.25% trypsin-EDTA, washed with PBS, detached with 0.25% trypsin-EDTA, and neutralized with growth medium. Following centrifugation for 5 minutes at 1,200 rpm, all cells, except HEY-A8 cells (PBS), were resuspended in either cold Hank's Balanced Salt Solution (HBSS; HCT 116 and A375) or cold HBSS and an equal volume of Matrigel Matrix. Cells were then injected in 100–200 μL volumes into either the right or left flanks of female nude mice, with the exception of MEL-JUSO cells, which were injected into SCID beige mice. Total cell numbers injected per mouse were 2 × 106 (HCT 116 and HEY-A8), 5 × 106 (MIA PaCa-2, HPAF-II, A375, Hs944.T, and SK-MEL-30), or 1 × 107 (MEL JUSO, Calu-6, and PC-3). Five to 7 mice were used for each group.
3990HPAX, 2043HPAX, 1290HCOX, 2861HCOX, 1855HCOX, 2094HLUX, and 3486HMEX patient-derived tumor xenograft tumors and the WM793 xenograft were propagated by serial passage of tumor fragments in nude mice. Briefly, 3 × 3 × 3 mm fragments of fresh tumor from a previous passage were implanted subcutaneously into the mice. Efficacy was carried with n = 6–12 mice per group.
Percent change in body weight (BW) was calculated as (BWcurrent − BWinitial)/(BWinitial) × 100%. Data were presented as mean percent body weight change from the day of treatment initiation ± SEM. Tumor volume was determined as published previously. All data were expressed as mean ± SEM. Changes in tumor volume were used for statistical analysis, and between-group comparisons were carried out using a one-way ANOVA. For all statistical evaluations, the level of significance was set at P < 0.05. Significance compared with the vehicle control group is reported, unless otherwise stated.

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Drug formulation [2]
Naporafenib (LXH-254) was dosed orally in MEPC4 vehicle [45% Cremophor RH40 + 27% PEG400 + 18% Corn Oil Glycerides (Maisine CC) + 10% ethanol] for all experiments, except PC3, where Naporafenib (LXH-254) was formulated in 90% PEG400 + 10% Tween80. MEPC4 stock was diluted 5 × (1:4 with de-ionized water) prior to dosing. Trametinib was dosed orally as a suspension in 0.5% HPMC and 0.2% Tween80 in distilled water at pH 8. Trametinib was stirred overnight and protected from light at room temperature prior to dosing.

Tolerability and Safety
All patients discontinued study treatment. The primary reason was disease progression (PD) (Supplementary Table 3). In naproxenfenib dose escalation and naproxenfenib/spartalizumab dose escalation and extension therapy, 14 (16.1%; due to adverse events: 3 [3.4%]), 6 (50.0%; due to adverse events: 5 [41.7%]), and 20 (46.5%; due to adverse events: 10 [23.3%]) patients reported at least one naproxenfenib dose reduction, respectively; and 87 (100%; due to adverse events: 34 [39.1%]), 12 (100%; due to adverse events: 9 [75.0%]), and 42 (97.7%; due to adverse events: 20 [46.5%]) patients reported at least one naproxenfenib dose interruption, respectively. In the monotherapy group, 5 out of 68 patients (7.4%) experienced at least one dose-limiting toxicity (DLT): grade 4 thrombocytopenia (1 patient; 1200 mg once daily), grade 3 neuralgia and grade 3 maculopapular rash/grade 3 pruritus (2 patients; 600 mg twice daily), and grade 3 bilirubin elevation/grade 3 hyponatremia and grade 3 peripheral sensory neuropathy (2 patients; 800 mg twice daily). The maximum tolerated dose (MTD) for naproxenfenib monotherapy at the highest tested dose of 1200 mg in the once-daily dosing regimen was not formally established. The MTD/RDE for the twice-daily naproxenfenib monotherapy regimen was formally established at 600 mg. Given that the once-daily and twice-daily dosing regimens were expected to have the same safety profile (depending on relative dose intensity), the twice-daily dosing regimen was chosen over the once-daily dosing regimen to reduce the medication burden. In the combination therapy dose escalation group, the starting dose was lower than the maximum tolerated dose (MTD) (400 mg twice daily). No dose-limiting toxicities (DLTs) were reported after treatment with naproxenfenib 400 mg BID/spartalizumab or naproxenfenib 600 mg BID/spartalizumab. The MTD for naproxenfenib/spartalizumab has not been reached. Due to a safety concern (grade 3 rash) with naproxenfenib 600 mg BID/spartalizumab, the recommended dose (RDE) for naproxenfenib in combination therapy was determined to be 400 mg BID. In the monotherapy group, 79 patients (90.8%) reported any grade of treatment-related adverse events (TRAEs). The most common adverse events (incidence ≥20%) were acne-like dermatitis (maculopapular pustular) (21 cases [24.1%], no grade 3/4 adverse events), rash (21 cases [24.1%], grade 3/4: 1 case [1.1%]), and fatigue (18 cases [20.7%], grade 3/4: 2 cases [2.3%]) (Table 2, Supplementary Table 4). In dose-escalation therapy with naproxenfenib/spartalizumab, 11 patients (91.7%) reported any grade of treatment-related adverse events; adverse events with an incidence ≥20% were nausea and pruritus (4 cases each [33.3%], no grade 3/4 adverse events) and acne-like dermatitis (3 cases [25%], grade 3/4: 1 case [8.3%]) (Table 2, Supplementary Table 5). During dose expansion therapy, 39 patients (90.7%) reported any grade of treatment-related adverse events (TRAEs). The most common TRAE was rash (any grade: 17 cases [39.5%], grade 3/4: 6 cases [14.0%]) (Table 2, Supplementary Table 5). Treatment-related adverse events reported at different dose levels and regimens are detailed in Supplementary Tables 6 and 7, and treatment-related serious adverse events are detailed in Supplementary Tables 8 and 9. Grade 3 neurological adverse events associated with naproxenfenib (occurring during interruption of naproxenfenib monotherapy or at the end of study treatment) included 2 cases of neuropathic pain, 1 case of peripheral sensory neuropathy, and 1 case of seizure; while 1 case of myalgia was reported in patients receiving naproxenfenib/spartalizumab combination therapy (data archive). During dose-escalation treatment, 11 patients (12.6%) in the monotherapy group died from primary malignancy, and 1 patient in the combination therapy group died from primary malignancy. In the dose-extension trial, 4 patients (9.3%) died (3 from underlying malignancy and 1 from COVID-19 infection). https://pmc.ncbi.nlm.nih.gov/articles/PMC11380116/#S12

The median time to peak concentration (Tmax) of naproxen was independent of dose and dosing regimen, and AUC0-last and Cmax increased approximately proportionally with increasing dose in both regimens (Supplementary Table 10). When naproxen was administered in combination with spartalizumab, the median Tmax, Cmax, and AUC0-last were 3.08–4.00 hours for the 400 mg BID regimen and 2.15 hours for the 600 mg BID regimen (Supplementary Table 11). On days 1 and 15, the geometric mean Cmax and AUC0-last of naproxen monotherapy with 400 mg BID and 600 mg BID were consistent with these values (Supplementary Table 11). Arithmetic mean concentration-time curves for all groups are shown in Supplementary Figure 2. A statistically significant association was observed between the naproxen Cmax range and the probability of developing grade ≥2 rash (Supplementary Figure 3). For details on the assessment of rBA and immunogenicity results, please refer to the supplemental data: https://pmc.ncbi.nlm.nih.gov/articles/PMC11380116/#S12

[1]. THERAPEUTIC COMBINATIONS COMPRISING A RAF INHIBITOR AND A ERK INHIBITOR. WO 2018051306 A1 20180322

[2]. LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors. Clin Cancer Res. 2021 Apr 1;27(7):2061-2073.

Napalafenib is an orally administered inhibitor of all members of the Raf family of serine/threonine protein kinases with potential antitumor activity. After administration, napalafenib binds to Raf proteins, inhibiting Raf-mediated signal transduction pathways, thereby suppressing the proliferation of Raf-overexpressing tumor cells. Raf protein kinases are key enzymes in the Ras/Raf/MEK/ERK signaling pathway and are upregulated in various cancer cell types. They play a crucial role in tumor cell proliferation and survival.

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Objective: Targeting RAF for antitumor therapy in RAS-mutant tumors holds great promise. This article details the novel properties of the type II RAF inhibitor napalafenib (LXH-254).Experimental Design: We performed biochemical, in vitro, and in vivo experimental analyses of napalafenib (LXH-254), including detecting the drug's activity in various cancer cell lines and a range of in vivo models. Furthermore, we evaluated the activity of naproxen (LXH-254) in cells with different RAF paralog knockout genes or in cells expressing ARAF variants with impaired kinase activity and dimer deficiency. Results: We found that naproxen (LXH-254) exhibits unexpected paralog selectivity, effectively inhibiting BRAF and CRAF, but with lower activity against ARAF. Naproxen (LXH-254) is active in models carrying BRAF alterations, including atypical BRAF alterations co-expressed with mutant K/NRAS and NRAS mutants, but with weaker activity in KRAS mutants. In RAS mutant cell lines, the absence of ARAF (rather than BRAF or CRAF) sensitizes cells to LXH254. ARAF-mediated LXH254 resistance requires kinase function and dimerization. Compared with RAS mutant cells expressing BRAF or CRAF, RAS mutant cells expressing only ARAF require higher concentrations of LXH254 to inhibit signal transduction. In addition, in cells expressing only ARAF, LXH254 induces paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo experiments, LXH254 completely regresses the homolog of RAS mutant cells lacking ARAF expression, while parental cell lines show only slight sensitivity. Conclusion: LXH254 is a novel RAF inhibitor that can inhibit dimerized BRAF and CRAF as well as monomeric BRAF, while having little effect on ARAF. [2] Consistent with this hypothesis, we found that in the MIA PaCa-2 model, even the addition of a low dose of trametinib to the full dose of LXH254 improved antitumor activity, which was superior to monotherapy (Figure 7D). The observation that adult mice could tolerate the simultaneous absence of BRAF and CRAF (8) suggests that weaker ARAF inhibition compared to drugs such as MEK and ERK inhibitors may improve the tolerability of LXH254, thereby contributing to drug exposure levels where both BRAF and CRAF are potently inhibited. This improved tolerability may facilitate combination therapy with other MAPK inhibitors for the treatment of RAS-mutant tumors, thereby enhancing pathway inhibition and antitumor effects. Supporting this view is the superior tolerability/efficacy ratio of LXH254-anchored MAPK combination therapy in preclinical animal models, as shown in Figure 7 (paper in progress). Furthermore, a dose-expansion study is currently underway for LXH254 combination therapy with the MEK inhibitor trametinib and the ERK inhibitor LTT462 in patients with multiple RAS/RAF pathway alterations.

C25H25F3N4O4

502.4856

502.49

C, 59.76; H, 5.01; F, 11.34; N, 11.15; O, 12.74

1800398-38-2

1800398-38-2;LXH254 HCl;

90456533

Off-white to light yellow solid powder

3.2

2

10

7

36

709

0

FC(C1C=C(C=CN=1)C(NC1C=CC(C)=C(C=1)C1C=C(N=C(C=1)N1CCOCC1)OCCO)=O)(F)F

UEPXBTCUIIGYCY-UHFFFAOYSA-N

InChI=1S/C25H25F3N4O4/c1-16-2-3-19(30-24(34)17-4-5-29-21(12-17)25(26,27)28)15-20(16)18-13-22(32-6-9-35-10-7-32)31-23(14-18)36-11-8-33/h2-5,12-15,33H,6-11H2,1H3,(H,30,34)

N-[3-[2-(2-hydroxyethoxy)-6-morpholin-4-ylpyridin-4-yl]-4-methylphenyl]-2-(trifluoromethyl)pyridine-4-carboxamide

LXH-254; Naporafenib; LXH254; LXH254; Naporafenib; 1800398-38-2; Naporafenib [INN]; N-(3-(2-(2-hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide; LXH254 free base; Pan-raf inhibitor LXH254; LXH 254

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~199.0 mM)
Ethanol: ~100 mg/mL (~199.0 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.98 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.98 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.98 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2.5 mg/mL (4.98 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)